Abstract In 2017, the WHO designated carbapenem-resistant Enterobacteriaceae (CRE) a Priority 1 ?critical superbug?. As few therapies remain to treat CRE, the risk of ?pan-resistant? CRE untreatable by any currently available antibiotic also exists, and entirely new agents with novel mechanisms of action (MOA) not cross-resistant to SOC agents languish. We believe that in addition to new strategies to treat CRE infections, greater consideration should also be given to preventing CRE infections from occurring in the first place. Our proposal aims to develop an O-antigen (O-a) biosynthetic inhibitory agent that potentiates serum-mediated killing (SMK) and is efficacious in prophylactic CRE rodent models of infection. We have shown that the O-a biosynthesis gene wecA, a nonessential gene under standard growth conditions, is essential for growth and pathogenesis in the presence of mammalian serum. Our proposal outlines a plan to develop synthetic inhibitors of WecA that we previously discovered and optimized to inhibit the Gram-positive WecA ortholog, TarO. Our Aims are: Aim 1. MOA validation and assay development. Demonstrate tarocins directly target WecA to elicit SMK effects. Establish target engagement (TE) and pathway engagement (PE) screening assays. Build isogenic set of permeability and efflux deficient mutants in E. coli (Ec), K. pneumoniae (Kp), P. aeruginosa (Pa), and A. baumannii (Ab) to guide med chem SAR. Milestone (M) 1. Identify up to 4 chemically distinct tarocin subseries for progression to Lead ID with > 4-fold EC50 shift in SMK by WecA overexpression, dose- dependent depletion of O-a, and causal drugR mutations mapping to wecA. Aim 2. Lead Identification. Drive SAR efforts to achieve WT activity by empirically assaying for SMK against a panel of Ec/Kp/Pa/Ab permeability and efflux deficient mutants, employing the emerging ?Hergenrother physicochemical rules of GN entry?, and exploring the utility of siderophore conjugation. M2. Identify up to 2 distinct tarocin subseries for progression to Lead Opt with Ec and Kp WT activity (Human Serum (HS)-MIC < 16 ug/ml), exposure to cover HS-MIC for 4 hrs, Ec TE and PE selectivity, Ec and Kp FOR < 1 x 10-9 at 8X HS- MIC, > 90% mammalian cell viability at 10 X HS-MIC, and HS-MIC90 < 64 ug/ml (n=50 isolates). Aim 3. Lead Optimization. Optimize PK, drug-like properties, and in vitro activity. M3. Identify 3 tarocin analogs for progression to Aim 4 with Ec/Kp WT activity (HS-MIC < 1 ug/ml), exposure to cover HS-MIC 8 hrs, Ec TE and PE selectivity, Ec/Kp FOR 1x 10-9 at 4X HS-MIC, >90% HepG2/HEK293 cell viability at 10X HS-MIC, clean CYP/ion channels (>10 uM), PanLabs IC50 > 10 uM, and HS-MIC90 2 ug/ml (n=100 isolates). Aim 4. In vivo efficacy demonstration. Demonstrate efficacy with up to three lead optimized compounds in rodent prophylaxis models of CRE bacteremia and thigh infections. M4. Identify 1 tarocin analog as a pre- clinical prophylaxis candidate that demonstrates > 3 log reduction of bacterial burden after 24h treatment in prophylactic rat CRE bacteremia and thigh infection models.